The long-term objective of the project is to understand the biochemical mechanisms of action of experimentally useful growth-inhibitory agents with therapeutic potential as targeted tumor therapies. The specific aims of this proposal are to identify the molecular targets, biochemical mechanisms of action and potential synergistic interactions of two compounds, and structurally related analogs, that up-regulate cell-cell communication, inhibit growth and reverse the transformed phenotype in ras-transformed tumorigenic cells, human lung tumor cells, or tumor promoter-treated cells at non-cytotoxic concentrations. Both compounds show preferential growth inhibition of transformed cells over non-transformed cells. One of these compounds, 4-phenyl-3-buteonic acid (PBA) modulates the p38 MAPK signaling pathway, up-regulates cell-cell communication and increases connexin phosphorylation in both ras-transformed epithelial cells and human lung tumor cells, as does its methyl ester form (PBA-Me) at 10-fold lower concentration. The other compound, chaetoglobosin K, modulates the PI- 3 kinase/Akt signaling pathway, inhibits cytokinesis in ras-transformed and src- transformed cells, and stabilizes cell-cell communication in tumor promoter-treated cells. The approach and methods to achieve these aims include use of phosphorylation site-specific antibodies that recognize activated or inhibited forms of kinases and phosphatases in western blot and ELISA assays, cell transfections with altered PI-3 kinase pathway and p38 MAPK genes and use of siRNAs to assist in identification of the molecular target of each compound, use of known p38 MAPK and PI-3 kinase pathway inhibitors and in vitro kinase activity assays to determine specific mechanisms of inhibition or activation, immunofluorescent staining to monitor intracellular protein localization, fluorescent dye-transfer assays to monitor cell-cell communication, and 1 and 2-D electrophoresis to monitor connexin phosphorylation changes at specific sites. Results from these studies will contribute further to our understanding of the role of gap junction-mediated cell-cell communication and the Akt kinase and p38 MAPK signaling pathways in tumor cell growth in vitro. In addition, elucidation of how these unique anti-tumor compounds act at a molecular level will allow assesment of their status as new prototype anti-tumor agents to treat human lung and other types of cancer. PUBLIC HEALTH RELEVANCE: Persistent activation of signaling pathways that control cell growth can lead to tumor formation. The proposed studies will investigate novel compounds which target specific growth-regulated cellular pathways known to be over-activated in many human cancers. Information obtained will allow assessment of these compounds as new prototype anti- tumor agents to treat lung and other types of cancer.